In Vitro Cross-Linking of Mycobacterium tuberculosis Peptidoglycan by L,D-Transpeptidases and Inactivation of These Enzymes by Carbapenems
In Vitro Cross-Linking of Mycobacterium tuberculosis Peptidoglycan by L,D-Transpeptidases and Inactivation of These Enzymes by Carbapenems
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DOI：
10.1128/aac.01663-13
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发表时间：
2013-12-01
影响因子：
4.9
通讯作者：
Mainardi, Jean-Luc
中科院分区：
文献类型：
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作者：
Cordillot, Mathilde;Dubee, Vincent;Mainardi, Jean-Luc
结核分枝杆菌肽聚糖主要通过L，D-转肽酶（LDT）交联，后者可被单一β-内酰胺类（碳青霉烯类）有效灭活。碳青霉烯类药物与β-内酰胺酶抑制剂克拉维酸联合应用，对广泛耐药的结核分枝杆菌具有一致的活性，因此，近年来碳青霉烯类药物治疗结核病的研究引起了人们的极大兴趣。结核病和杀死指数增长和休眠形式的杆菌。我们纯化了M.结核菌素（Mt 1 - 5）的活性，并与肽聚糖片段和碳青霉烯类抗生素的活性进行比较。5种LDT在体外具有功能性，因为它们在肽聚糖交联（Mt5）、β-内酰胺酰化（Mt3）或两者（Mt 1、Mt 2和Mt4）试验中具有活性。Mt3是唯一的LDT是在交联试验中无活性的，这表明这种酶可能参与其他细胞功能，如蛋白质锚定肽聚糖，如大肠杆菌中所示。碳青霉烯类对LDTs的灭活是一个两步反应，包括可逆形成四面体中间体（含氧阴离子），然后是β-内酰胺环的不可逆断裂，导致形成稳定的酰基酶。这两个步骤的速率常数的测定揭示了碳青霉烯类之间的重要差异（高达460倍），这影响了氧阴离子和酰基酶形成的速度。亚胺培南灭活LDTs比厄他培南更快，这两种药物比美罗培南和多利培南更有效，表明碳青霉烯侧链的修饰可用于优化其抗分枝杆菌活性。
The Mycobacterium tuberculosis peptidoglycan is cross-linked mainly by L,D-transpeptidases (LDTs), which are efficiently inactivated by a single beta-lactam class, the carbapenems. Development of carbapenems for tuberculosis treatment has recently raised considerable interest since these drugs, in association with the beta-lactamase inhibitor clavulanic acid, are uniformly active against extensively drug-resistant M. tuberculosis and kill both exponentially growing and dormant forms of the bacilli. We have purified the five L,D-transpeptidase paralogues of M. tuberculosis (Mt1 to -5) and compared their activities with those of peptidoglycan fragments and carbapenems. The five LDTs were functional in vitro since they were active in assays of peptidoglycan cross-linking (Mt5), beta-lactam acylation (Mt3), or both (Mt1, Mt2, and Mt4). Mt3 was the only LDT that was inactive in the cross-linking assay, suggesting that this enzyme might be involved in other cellular functions such as the anchoring of proteins to peptidoglycan, as shown in Escherichia coli. Inactivation of LDTs by carbapenems is a two-step reaction comprising reversible formation of a tetrahedral intermediate, the oxyanion, followed by irreversible rupture of the beta-lactam ring that leads to formation of a stable acyl enzyme. Determination of the rate constants for these two steps revealed important differences (up to 460-fold) between carbapenems, which affected the velocity of oxyanion and acyl enzyme formation. Imipenem inactivated LDTs more rapidly than ertapenem, and both drugs were more efficient than meropenem and doripenem, indicating that modification of the carbapenem side chain could be used to optimize their antimycobacterial activity.